Preparation,anticholinesterase activity and molecular docking of new lupane derivatives

A set of twenty one lupane derivatives (2–22) was prepared from the natural triterpenoid calenduladiol (1) by transformations on the hydroxyl groups at C-3 and C-16, and also on the isopropenyl moiety. The derivatives were tested for their inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and some structure–activity relationships were outlined with the aid of enzyme kinetic studies and docking modelization. The most active compound resulted to be 3,16,30-trioxolup-20(29)-ene (22), with an IC₅₀ value of 21.5 μM for butyrylcholinesterase, which revealed a selective inhibitor profile towards this enzyme.     

New derivatives of lupane triterpenoids disturb breast cancer mitochondria and induce cell death

Novel cationic dimethylaminopyridine derivatives of pentacyclic triterpenes were previously described to promote mitochondrial depolarization and cell death in breast and melanoma cell lines. The objective of this work was to further investigate in detail the mechanism of mitochondrial perturbations, correlating those effects with breast cancer cell responses to those same agents. Initially, a panel of tumor and non-tumor cell lines was grown in high-glucose or glucose-free glutamine-containing media, the later forcing cells to synthesize ATP by oxidative phosphorylation only. Cell proliferation, cell cycle, cell death and mitochondrial membrane polarization were evaluated. Inhibition of cell proliferation was observed, accompanied by an arrest in the G1-cell cycle phase, and importantly, by loss of mitochondrial membrane potential. On a later time-point, caspase-9 and 3 activation were observed, resulting in cell death. For the majority of test compounds, we determined that cell toxicity was augmented in the galactose media. To investigate direct evidences on mitochondria isolated rat liver mitochondria were used. The results showed that the compounds were strong inducers of the permeability transition pore. Confirming our previous results, this work shows that the novel DMAP derivatives strongly interact with mitochondria, resulting in pro-apoptotic signaling and cell death.     

alpha,beta-Dehydrophenylalanine choline esters, a new class of reversible inhibitors of human acetylcholinesterase and butyrylcholinesterase

Our goal was to design, synthesize, and evaluate new cholinesterase inhibitors. Fourteen dehydroamino acids esterified to choline and to its ternary analog were synthesized by a new method that gave a yield of 84-93%. The potency of the amino acid ester derivatives was tested by measuring K(i) values for inhibition of human red cell acetylcholinesterase and human plasma butyrylcholinesterase. The most potent compound was a choline ester of dehydrophenylalanine where the amine group of the amino acid was derivatized with a benzoyl group containing a methoxy in the 2-position, CH(3)O(C(6)H(4))CONHC(CHC(6)H(5))COOCH(2)CH(2)N(+)(CH(3))(3). This compound was a strong inhibitor of both human acetylcholinesterase and human butyrylcholinesterase, with K(i) values of 10 microM and 0.08 microM, respectively. These K(i) values are comparable to that of Rivastigmine. Docking of the most potent compound into the active site of human butyrylcholinesterase showed that the lowest energy model had two benzene rings oriented towards Trp 82 and Tyr 332 whereas the positively charged nitrogen group was stabilized by Trp 231. This orientation placed the ester group 3.89 A from the active site Ser 198, a distance too far for covalent bonding, explaining why the esters are inhibitors rather than substrates. This class of anticholinesterase agents has the potential for therapeutic utility in the treatment of disorders of the cholinergic system.     

Synthesis and cholinesterase inhibition of cativic acid derivatives

Alzheimer’s disease (AD) is a neurodegenerative disorder associated with memory impairment and cognitive deficit. Most of the drugs currently available for the treatment of AD are acetylcholinesterase (AChE) inhibitors. In a preliminary study, significant AChE inhibition was observed for the ethanolic extract of Grindelia ventanensis (IC₅₀ = 0.79 mg/mL). This result prompted us to isolate the active constituent, a normal labdane diterpenoid identified as 17-hydroxycativic acid (1), through a bioassay guided fractionation. Taking into account that 1 showed moderate inhibition of AChE (IC₅₀ = 21.1 μM), selectivity over butyrylcholinesterase (BChE) (IC₅₀ = 171.1 μM) and that it was easily obtained from the plant extract in a very good yield (0.15% w/w), we decided to prepare semisynthetic derivatives of this natural diterpenoid through simple structural modifications. A set of twenty new cativic acid derivatives (3–6) was prepared from 1 through transformations on the carboxylic group at C-15, introducing a C2–C6 linker and a tertiary amine group. They were tested for their inhibitory activity against AChE and BChE and some structure–activity relationships were outlined. The most active derivative was compound 3c, with an IC₅₀ value of 3.2 μM for AChE. Enzyme kinetic studies and docking modeling revealed that this inhibitor targeted both the catalytic active site and the peripheral anionic site of this enzyme. Furthermore, 3c showed significant inhibition of AChE activity in SH-SY5Y human neuroblastoma cells, and was non-cytotoxic.     

Cyclic acyl guanidines bearing carbamate moieties allow potent and dirigible cholinesterase inhibition of either acetyl- or butyrylcholinesterase

A series of cyclic acyl guanidine with carbamate moieties have been synthesized and evaluated in vitro for their AChE and BChE inhibitory activities. Structure−activity relationships identified compound 23 as a nanomolar and selective BChE inhibitor, while compound 32 exhibited nanomolar and selective AChE inhibition, selectivity depending on both the structure of the carbamate substituent as well as the position of guanidines-N substitution. The velocity of enzyme carbamoylation was analyzed and showed similar behavior to physostigmine. Phenolic compounds formed after carbamate transfer to the active site of cholinesterases showed additional neuroprotective properties on a hippocampal neuronal cell line (HT-22) after glutamate-induced intracellular reactive oxygen species generation.     

Design,synthesis and evaluation of novel 7-aminoalkyl-substituted flavonoid derivatives with improved cholinesterase inhibitory activities

A novel series of 7-aminoalkyl-substituted flavonoid derivatives 5a–5r were designed, synthesized and evaluated as potential cholinesterase inhibitors. The results showed that most of the synthesized compounds exhibited potent acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities at the micromolar range. Compound 2-(naphthalen-1-yl)-7-(8-(pyrrolidin-1-yl)octyloxy)-4H-chromen-4-one (5q) showed the best inhibitory activity (IC₅₀, 0.64 μM for AChE and 0.42 μM for BChE) which were better than our previously reported compounds and the commercially available cholinergic agent Rivastigmine. The results from a Lineweaver–Burk plot indicated a mixed-type inhibition for compound 5q with AChE and BChE. Furthermore, molecular modeling study showed that 5q targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Besides, these compounds (5a–5r) did not affect PC12 and HepG2 cell viability at the concentration of 10 μM. Consequently, these flavonoid derivatives should be further investigated as multipotent agents for the treatment of Alzheimer’s disease.     

Insights into (S)-rivastigmine inhibition of butyrylcholinesterase (BuChE): Molecular docking and saturation transfer difference NMR (STD-NMR)

Rivastigmine is a very important drug prescribed for the treatment of Alzheimer’s disease (AD) symptoms. It is a dual inhibitor, in that it inhibits both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). For our screening program on the discovery of new rivastigmine analogue hits for human butyrylcholinesterase (hBuChE) inhibition, we investigated the interaction of this inhibitor with BuChE using the complimentary approach of the biophysical method, saturation transfer difference (STD)-NMR and molecular docking. This allowed us to obtain essential information on the key binding interactions between the inhibitor and the enzyme to be used for screening of hit compounds. The main conclusions obtained from this integrated study was that the most dominant interactions were (a) H-bonding between the carbamate carbonyl of the inhibitor and the NH group of the imidazole unit of H434, (b) stacking of the aromatic unit of the inhibitor and the W82 aromatic unit in the choline binding pocket via π-π interactions and (c) possible CH/π interactions between the benzylic methyl group and the N-methyl groups of the inhibitor and W82 of the enzyme.     

Design,synthesis and biological evaluation of benzofuran appended benzothiazepine derivatives as inhibitors of butyrylcholinesterase and antimicrobial agents

A series of bezofuran appended 1,5-benzothiazepine compounds 7a–v was designed, synthesized and evaluated as cholinesterase inhibitors. The biological assay experiments showed that most of the compounds displayed a clearly selective inhibition for butyrylcholinesterase (BChE), while a weak or no effect towards acetylcholinesterase (AChE) was detected. All analogs exhibited varied BChE inhibitory activity with IC₅₀ value ranging between 1.0?±?0.01 and 72?±?2.8?μM when compared with the standard donepezil (IC₅₀, 2.63?±?0.28?μM). Among the synthesized derivatives, compounds 7l, 7m and 7k exhibited the highest BChE inhibition with IC₅₀ values of 1.0, 1.0 and 1.8?μM, respectively. The results from a Lineweaver-Burk plot indicated a mixed-type inhibition for compound 7l with BChE. In addition, docking studies confirmed the results obtained through in vitro experiments and showed that most potent compounds bind to both the catalytic anionic site (CAS) and peripheral anionic site (PAS) of BChE active site. The synthesized compounds were also evaluated for their in vitro antibacterial and antifungal activities. The results indicated that the compounds possessed a broad spectrum of activity against the tested microorganisms and showed high activity against both gram positive and gram negative bacteria and fungi.     

Synthesis and in vitro evaluation of 7-methoxy-N-(pent-4-enyl)-1,2,3,4-tetrahydroacridin-9-amine-new tacrine derivate with cholinergic properties

Cholinesterase inhibitors are, so far, the only successful strategy for the symptomatic treatment of Alzheimer's disease. Tacrine (THA) is a potent acetylcholinesterase inhibitor that was used in the treatment of Alzheimer's disease for a long time. However, the clinical use of THA was hampered by its low therapeutic index, short half-life and liver toxicity. 7-Methoxytacrine (7-MEOTA) is equally pharmacological active compound with lower toxicity compared to THA. In this Letter, the synthesis, biological activity and molecular modelling of elimination by-product isolated during synthesis of 7-MEOTA based bis-alkylene linked compound is described.     

Synthesis and characterization of new inhibitors of cholinesterases based on N-phenylcarbamates: In vitro study of inhibitory effect,type of inhibition,lipophilicity and molecular docking

Based on current treatment of Alzheimer's disease, where the carbamate inhibitor Rivastigmine is used, two series of carbamate derivatives were prepared: (i) N-phenylcarbamates with additional carbamate group (1–12) and (ii) N-phenylcarbamates with monosaccharide moiety (13–24). All compounds were tested for the inhibitory effect on both of the cholinesterases, electric eel acetylcholinesterase (eeAChE) and butyrylcholinesterase from equine serum (eqBChE) and the inhibitory activity (expressed as IC₅₀ values) was compared with that of the established drugs Galanthamine and Rivastigmine. The compounds with two carbamate groups 1–12 revealed higher inhibitory efficiency on both cholinesterases in compared with monosaccharide derived carbamates 13–24 and with Rivastigmine. The significant decrease of inhibitory efficiency on eqBChE (also for eeAChE but in less manner) was observed after deacetalization of monosaccharide. Moreover, the type of inhibitory mechanism of five chosen compounds was studied. It was found, that compounds with two carbamate groups act presumably via a mixed inhibitory mechanism and the compounds with monosaccharide moiety act as non-competitive inhibitors. The lipophilicity of tested compounds was determined using partition coefficient. Specific positions of the inhibitors in the binding sites of cholinesterases were determined using molecular modeling and the results indicate the importance of phenylcarbamate orientation in the catalytic gorges of both enzymes.     

Exploring new chemical functionalities to improve aromatase inhibition of steroids

In this work, new potent steroidal aromatase inhibitors both in microsomes and in breast cancer cells have been found. The synthesis of the 3,4-(ethylenedioxy)androsta-3,5-dien-17-one (12), a new steroid containing a heterocycle dioxene fused in the A-ring, led to the discovery of a new reaction for which a mechanism is proposed. New structure–activity relationships were established. Some 5β-steroids, such as compound 4β,5β-epoxyandrostan-17-one (9), showed aromatase inhibitory activity, because they adopt a similar A-ring conformation as those of androstenedione, the natural substrate of aromatase. Moreover, new chemical features to increase planarity were disclosed, specifically the 3α,4α-cyclopropane ring, as in 3α,4α-methylen-5α-androstan-17-one (5) (IC₅₀ = 0.11 μM), and the Δ^9–11 double bond in the C-ring, as in androsta-4,9(11)-diene-3,17-dione (13) (IC₅₀ = 0.25 μM). In addition, induced-fit docking (IFD) simulations and site of metabolism (SoM) predictions helped to explain the recognition of new potent steroidal aromatase inhibitors within the enzyme. These insights can be valuable tools for the understanding of the molecular recognition process by the aromatase and for the future design of new steroidal inhibitors.     

In vitro and in silico evaluation of new thiazole compounds as monoamine oxidase inhibitors

New twenty compounds bearing thiazole ring (3a-3t) were designed and synthesized as monoamine oxidase (MAO) inhibitors. The fluorometric enzyme inhibition assay was used to determine the biological effects of synthesized compounds. Most of them showed remarkable inhibitory activity against both MAO-A and MAO-B. By comparing their IC₅₀ values, it can be seen that active derivatives displayed generally selectivity on MAO-B enzyme. Compounds 3j and 3t, which bear dihydroxy moiety at the 3rd and 4th position of phenyl ring, were the most active derivatives in the series against both isoenzymes. Compounds 3j and 3t showed significant inhibition profile on MAO-A with the IC₅₀ values of 0.134 ± 0.004 µM and 0.123 ± 0.005 µM, respectively, while they performed selectivity against MAO-B with the IC₅₀ values of 0.027 ± 0.001 µM and 0.025 ± 0.001 µM, respectively. Also, docking studies about these compounds were carried out to evaluate their binding modes on the active regions of MAO-A and MAO-B.     

Synthesis,biological evaluation and molecular modeling of novel selective COX-2 inhibitors: sulfide,sulfoxide, and sulfone derivatives of 1,5-diarylpyrrol-3-substituted scaffold

A novel series of 1,5-diarylpyrrol-3-sulfur derivatives (10–12) was synthesized and characterized by NMR and mass spectroscopy and x-ray diffraction. The biological activity of these compounds was evaluated in in vitro and in vivo tests to assess their COX-2 inhibitory activity along with anti-inflammatory and antinociceptive effect.Results showed that the bioisosteric transformation of previously reported alkoxyethyl ethers (9a-c) into the corresponding alkyl thioethers (10a-c) still leads to selective and active compounds being the COX-2 inhibitory activity for most of them in the low nanomolar range. The oxidation products of 10a,b were also investigated and both couple of sulfoxides (11a,b) and sulfones (12a,b) showed an appreciable COX-2 inhibitory activity. Molecular modeling studies were performed to investigate the binding mode of the representative compounds 10b, 11b, and 12b into COX-2 enzyme and to explore the potential site of metabolism of 10a and 10b due to the different in vivo efficacy. Among the developed compounds, compound 10b showed a significant in vivo anti-inflammatory and antinociceptive activity paving the way to develop novel anti-inflammatory drugs.     

Synthesis, dihydrofolate reductase inhibition, antitumor testing, and molecular modeling study of some new 4(3H)-quinazolinone analogs

In order to produce potent new leads for anticancer drugs, a new series of quinazoline analogs was designed to resemble methotrexate (MTX, 1) structure features and fitted with functional groups believed to enhance inhibition of mammalian DHFR activity. Molecular modeling studies were used to assess the fit of these compounds within the active site of human DHFR. The synthesized compounds were evaluated for their ability to inhibit mammalian DHFR in vitro and for their antitumor activity in a standard in vitro tissue culture assay panel. Compounds 28, 30, and 31 were the most active DHFR inhibitors with IC₅₀ values of 0.5, 0.4, and 0.4 μM, respectively. The most active antitumor agents in this study were compounds 19, 31, 41, and 47 with median growth inhibitory concentrations (GI₅₀) of 20.1, 23.5, 26.7, and 9.1 μM, respectively. Of this series of compounds, only compound 31 combined antitumor potency with potent DHFR inhibition; the other active antitumor compounds (19, 41, and 47) all had DHFR IC₅₀ values above 15 μM, suggesting that they might exert their antitumor potency through some other mode of action. Alternatively, the compounds could differ significantly in uptake or concentration within mammalian cells.     

Design,synthesis and anti-diabetic activity of triazolotriazine derivatives as dipeptidyl peptidase-4 (DPP-4) inhibitors

Type 2 diabetes mellitus (T2DM) is one of the major global metabolic disorders characterized by insulin resistance and chronic hyperglycemia. Inhibition of the enzyme, dipeptidyl peptidase-4 (DPP-4) has been proved as successful and safe therapy for the treatment of T2DM since last decade. In order to design novel DPP-4 inhibitors, various in silico studies such as 3D-QSAR, pharmacophore modeling and virtual screening were performed and on the basis of the combined results of them, total 50 triazolo[5,1-c][1,2,4]triazine derivatives were designed and mapped on the best pharmacophore model. From this, best 25 derivatives were docked onto the active site of DPP-4 enzyme and in silico ADMET properties were also predicted. Finally, top 17 derivatives were synthesized and characterized using FT-IR, Mass, ¹H NMR and ¹³C NMR spectroscopy. Purity of compounds was checked using HPLC. These derivatives were then evaluated for in vitro DPP-4 inhibition. The most promising compound 15q showed 28.05 μM DPP-4 IC₅₀ with 8–10-fold selectivity over DPP-8 and DPP-9 so selected for further in vivo anti-diabetic evaluation. During OGTT in normal C57BL/6J mice, compound 15q reduced blood glucose excursion in a dose-dependent manner. Chronic treatment for 28 days with compound 15q improved the serum glucose levels in type 2 diabetic Sprague Dawley rats wherein diabetes was induced by high fat diet and low dose streptozotocin. This suggested that compound 15q is a moderately potent and selective hit molecule which can be further optimized structurally to increase the efficacy and overall pharmacological profile as DPP-4 inhibitor.     

Identification of the stereochemical requirements in the 4-aryl-2-cycloalkylidenhydrazinylthiazole scaffold for the design of selective human monoamine oxidase B inhibitors

Exploring the effect that substituents on the cycloaliphatic ring had on the inhibitory activity against human monoamine oxidase B of a series of 4-aryl-2-cycloalkylidenhydrazinylthiazoles led to the synthesis of a new series of 2-methylcyclopentyl and 3-methylcyclopentyl derivatives which were tested in vitro as mixtures of diastereoisomers. In fact, due to the presence of a chiral center on the cycloaliphatic ring and a trisubstituted CN bond, they exist as four diastereoisomers ((E)-(R), (E)-(S), (Z)-(R), (Z)-(S)). 4-(2,4-Difluorophenyl)-2-(2-(3-methylcyclopentylidene)hydrazinyl)thiazole was chosen as a model to investigate the influence of stereochemical requirements on the inhibitory activity against hMAO-B of these derivatives after a stereoconservative synthesis and semi-preparative HPLC diastereoseparation. (R)-(Z) isomer of this compound was endowed with a potent and selective hMAO-B inhibition higher than that of reference drugs as also corroborated by molecular modeling studies.     

Novel oxazolinyl derivatives of pregna-5,17(20)-diene as 17α-hydroxylase/17,20-lyase (CYP17A1) inhibitors

New oxazolinyl derivatives of [17(20)E]-pregna-5,17(20)-diene: 2′-{[(E)-3β-hydroxyandrost-5-en-17-ylidene]methyl}-4′,5′-dihydro-1′,3′-oxazole 1 and 2′-{[(E)-3β-hydroxyandrost-5-en-17-ylidene]methyl}-4′,4′-dimethyl-4′,5′-dihydro-1′,3′-oxazole 2 were evaluated as potential CYP17A1 inhibitors in comparison with 17-(pyridin-3-yl)androsta-5,16-dien-3β-ol 3 (abiraterone). Differential absorption spectra of human recombinant CYP17A1 in the presence of compound 1 (λ_max = 422 nm, λ_min = 386 nm) and compound 2 (λ_max = 416 nm) indicated significant differences in enzyme/inhibitors complexes. CYP17A1 activity was measured using electrochemical methods. Inhibitory activity of compound 1 was comparable with abiraterone 3 (IC₅₀ = 0.9 ± 0.1 μM, and IC₅₀ = 1.3 ± 0.1 μM, for compounds 1 and 3, respectively), while compound 2 was found to be weaker inhibitor (IC₅₀ = 13 ± 1 μM). Docking of aforementioned compounds to CYP17A1 revealed that steroid fragments of compound 1 and abiraterone 3 occupied close positions; oxazoline cycle of compound 1 was coordinated with heme iron similarly to pyridine cycle of abiraterone 3. Configuration of substituents at 17(20) double bond in preferred docked position corresponded to Z-isomers of compounds 1 and 2. Presence of 4′-substituents in oxazoline ring of compound 2 prevents coordination of oxazoline nitrogen with heme iron and worsens its docking score in comparison with compound 1. These data indicate that oxazolinyl derivative of [17(20)E]-pregna-5,17(20)-diene 1 (rather than 4′,4′-dimethyl derivative 2) may be considered as potential CYP17A1 inhibitor and template for development of new compounds affecting growth and proliferation of prostate cancer cells.     

A technique to study molecular recognition in drug design: Preliminary application of free energy derivatives to inhibition of a malarial cysteine protease

We present molecular dynamics studies on model complexes of inhibitors of a malarial cysteine protease. The initial model for such complexes came from the model building of the protein using its homology with other cysteine proteases and calculations using DOCK to generate new lead compounds. Some of the initial model-built structures were quite stable for 100 psec of dynamics; others moved significantly from their model-built orientation. We also calculated the free energy derivatives at each atom in the inhibitor, both in water and in the binding site. The results of these calculations suggest directions for the design of new, more potent enzyme inhibitors and agree qualitatively with some of the experimental findings. Nonetheless, we stress that we have only used this methodology in an interpretive rather than a predictive manner.     

分子动力学模拟HPV(16, 18)E6蛋白

目的:对人乳头瘤病毒HPV16,18中E6蛋白结构进行分子模拟和分析,寻找可以作为蛋白-配体相互作用的关键结构区域。方法:以HPV16 E6蛋白为模板,对HPV18 E6蛋白进行同源建模,对构建的HPV18 E6模型以及晶体结构模型HPV16 E6进行分子动力学模拟,通过微观上的loop环分析和宏观上的整体运动分析研究了HPV18 E6与HPV16 E6在溶剂环境下结构变化的异同。结果:发现靠近N端loop环在蛋白-配体结合过程中能介导控制配体、水、离子进出的"门控"的作用,解释了两个蛋白在水溶剂中的运动构象的变化。结论:本研究解释了HPV16 E6与HPV18 E6两个蛋白在溶剂中的运动机制,并发现了loop环在其中扮演"门控"的作用,解释了两个蛋白在水溶剂中的运动构象的变化,该发现能够为基于两个蛋白为靶点的药物设计提供理论依据。